Force-dependent vinculin binding to talin in live cells: a crucial step in anchoring the actin cytoskeleton to focal adhesions
Force-dependent vinculin binding to talin in live cells: a crucial step in anchoring the actin cytoskeleton to focal adhesions
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DOI：
10.1152/ajpcell.00122.2013
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发表时间：
2014-03-01
影响因子：
5.5
通讯作者：
Sokabe, Masahiro
中科院分区：
文献类型：
--
作者：
Hirata, Hiroaki;Tatsumi, Hitoshi;Sokabe, Masahiro
机械力在局部粘连(FAs)的调节中起着关键作用，在FAs中，肌动蛋白细胞骨架通过整合素和包括talin和vinculin在内的各种连接蛋白锚定在细胞外基质上。纽蛋白在FAs的定位依赖于机械力。虽然体外研究已经证明了力诱导的纽蛋白与Talin结合的增加，但在体内FAs中是否存在这样的机制尚不清楚。在这项研究中，使用在弹性硅胶基质上培养的成纤维细胞，我们研究了力在调节FA上的Talin-vinculin结合中的作用。拉伸底物导致在含有talin的FA处纽蛋白的积累，这种积累通过表达vinculin的talin结合结构域(结构域d1，它抑制内源性vinculin与talin的结合)而被取消。这些结果表明，加载到FA上的机械力促进了FA上的纽蛋白与talin的结合。在细胞突起区域，肌动蛋白网络在表达D1域的细胞中向后移动到含有talin的FA上，而在对照细胞中它被锚定到FA上，这表明在活细胞中，依赖于力的vinculin与talin结合是将肌动蛋白细胞骨架锚定到FA上的关键。
Mechanical forces play a pivotal role in the regulation of focal adhesions (FAs) where the actin cytoskeleton is anchored to the extracellular matrix through integrin and a variety of linker proteins including talin and vinculin. The localization of vinculin at FAs depends on mechanical forces. While in vitro studies have demonstrated the force-induced increase in vinculin binding to talin, it remains unclear whether such a mechanism exists at FAs in vivo. In this study, using fibroblasts cultured on elastic silicone substrata, we have examined the role of forces in modulating talin-vinculin binding at FAs. Stretching the substrata caused vinculin accumulation at talin-containing FAs, and this accumulation was abrogated by expressing the talin-binding domain of vinculin (domain D1, which inhibits endogenous vinculin from binding to talin). These results indicate that mechanical forces loaded to FAs facilitate vinculin binding to talin at FAs. In cell-protruding regions, the actin network moved backward over talin-containing FAs in domain D1-expressing cells while it was anchored to FAs in control cells, suggesting that the force-dependent vinculin binding to talin is crucial for anchoring the actin cytoskeleton to FAs in living cells.